Haynes Lab:Notebook/Engineering PC-TFs/2014/09/15: Difference between revisions
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'''Protein extraction from transfected cells''' | '''Protein extraction from transfected cells''' | ||
* | * Warmed media, PBS, Trypsin to 37°C. | ||
* Warmed protease inhibitor cocktail (protease arrest) and protein extraction buffer to room temperature. | |||
* Removed six well plate from incubator. Two wells (W1, W2) have transfected cells. | |||
* Aspirated off media from each well. | |||
* Washed first three wells with 1mL PBS (W1, W2, Negative control). | |||
* Aspirated off PBS. Added 0.5mL Trypsin. Incubated for 3 minutes at 37°C. | |||
* Added 5mL media to each well. Pipetted up and down to remove remaining adherent cells. | |||
* Transferred cells from each well into their own 14mL conical tube. | |||
* Pelleted cells by spinning at 1000 x g for 3 minutes. | |||
* Aspirated off supernatent leaving ~100μL covering the pellet. | |||
* Resuspended cells in tube by flicking. Placed these cells on ice when not in direct use for following steps. | |||
* Labeled three 1.5mL tubes (W1, W2, Neg). | |||
* Added 500μL protein extraction buffer to each tube. | |||
* Added 5μL protease inhibitor cocktail (100x) to each tube. Inverted to mix. | |||
* Aspirated 505μL combination mixture and dispensed into corresponding tube containing cell pellet. Pipetted up and down to mix. | |||
* Aspirated total buffer, protease inhibitor, resuspended cell pellet and dispensed back into corresponding 1.5mL tube. | |||
* Repeated for other two samples. | |||
* Centrifuged samples at max rpm for five minutes in cold room. | |||
* Labeled three new 1.5mL tubes. | |||
* Transferred volume to new tube being careful not to disturb the pellet at the bottom of each tube. | |||
Revision as of 11:28, 16 September 2014
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Summary
Protein extraction from transfected cells
Plate reader measurements RFP signal
Standard curve
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